AIMS: To determine the reason for drug and xenobiotic specificity of the CYP enzymes in humans. The CYP2C subfamily is highly homologous at the gene level, but differ markedly in their substrate specificity. CYP2C9 specifically metabolizes a number of anti-inflammatory drugs such as ibuprofen and diclofenac. In contrast, the closely related CYP2C19 specifically metabolizes other drugs such as the anticonvulsant mephenytoin. The aims of this study are to define the amino acids involved in substrate specificity and solve the structure of these CYP2Cs. Structure activity studies will help predict which drugs will be metabolized by these polymorphic enzymes. ACCOMPLISHMENTS: We used mephenytoin as a prototype substrate for human CYP2C19. Preliminary experiments with chimeras between CYP2C9 and CYP2C19 indicated that multiple substrate recognition sites are necessary for CYP2C19 to be able to recognize and metabolize specific substrates Preliminary experiments suggest that I99H in a substrate recognition site (SRS) (SRS1) proposed by Gotoh in combination with 220Ser Pro and 220Pro Thr in the FG loop and SRS 3 and 14 of 2C19 are required to convert CYP2C9 to a high turnover mephenytoin hydroxylase. Later site directed mutagenesis experiments indicate that I99H, S221P, P221T, and three amino acids in SRS are required to convert CYP2C9 to a mephenytoin hydroxylase. These are 286, 2292, and 295. Therefore multiple regions appear to be required for CYP2C19 specificity, although conversely, only a few changes are required for to convert CYP2C19 to a 2C9 like enzyme. Substrate specificity with pesticides and environmental chemicals indicate that certain organophosphorus pesticides such as chlorpyrofos are metabolized at least in part by CYP2C19.